HAART is an effective treatment for HIV infection but can cause serious metabolic complications including lacticacidemia, lipodystrophy, peripheral neuropathies, and cardiomyopathies. There is considerable evidence that the metabolic complications are the result of unintended mitochondrial toxicity of HAART. Because these complications can limit treatment, there are pressing needs: 1) to better understand the molecular basis of this toxicity, and 2) for diagnostic tools to monitor the resulting mitochondrial dysfunction. Addressing both needs could provide the rationale and means to guide therapy so HAART could be modified before serious clinical complications ensue. To this end, we propose to make a set of simple dipstick-based immunoassays that will be used first in research directed at understanding the effects of therapy on key mitochondrial enzymes of energy metabolism, and then developed into point of care diagnostics. The enzymes measured constitute the oxidative phosphorylation system (OXPHOS) responsible for providing almost all cellular ATP. Not only are these enzyme complexes essential for mitochondrial function, but they are known to be affected in HAART as a result of the depletion of mtDNA. We have shown that depletion of OXPHOS complexes can be used as a surrogate for mtDNA depletion, as mtDNA encodes 13 protein subunits that are essential components of 4 of the 5 complexes of the OXPHOS system. Moreover, it is the levels of functional OXPHOS enzymes rather than mtDNA levels that most accurately reflects mitochondrial dysfunction Finally, the dipstick assays, unlike real time PCR used to measure mtDNA depletion directly, are simple quick, inexpensive and capable of high throughput. Our prototype Complex I specific dipsticks can detect Complex I in samples containing as little as 0.1ug of mitochondria or as few as 1x105 solubilized whole cells (fibroblasts or peripheral blood mononuclear cells). The prototype dipsticks can also detect the effects of mtDNA-depletion, i.e., that mtDNA-depleted human fibroblasts lack Complex I. We will examine 2 different ways to quantify protein levels with the dipsticks, 1 instrument-free using only inexpensive self reporting disposable dipsticks, the second incorporating an inexpensive dipstick reader to provide greater Decision. The tests will be optimized for sensitivity, accuracy, reliability and dynamic range, using cultured human fibroblasts and control samples from clinically relevant tissues, such as PBMCs and subcutaneous adipose tissue.